Functional Magnetic Resonance Imaging (fMRI) has become a method of choice for human functional neuroimaging studies, and is beginning to make inroads into clinical applications such as monitoring of stroke rehabilitation. However, in current practice, fMRI suffers from the uncertain relation of the imaged hemodynamic responses to the underlying neuroglial activity and quantitative hemodynamic parameters. Interpretation of fMRI studies in disease states are even more ambiguous since it requires not only understanding the mechanisms of neurovascular coupling but the impact of altered cerebrovascular dynamics on the BOLD signal under conditions of neurovascular deficit. As a result, the analysis of fMRI data has so far been largely correlational and descriptive. In order to gain a mechanistic understanding of the relationship between BOLD contrast and the underlying neuroglial activity, one has to consider multiple physiological processes, from macroscopic hemodynamic changes to the microscopic neurovascular communication. To this end, we will combine direct and quantitative measurement of hemodynamic and neuronal parameters simultaneously with fMRI aiming to understand the relationship between the BOLD response and the underlying neuroglial activity. We then will establish microscopic correlates of large-scale (populational) hemodynamic and neuroglial signals and will integrate macro- and microscopic measurements. Specifically, in Aim1 we will characterize BOLD signals in hemodynamic terms by employing simultaneous optical imaging of blood oxygenation and blood flow. In Aim2 we will establish a correlation between stimulus-induced BOLD response and the underlying neuronal and astrocytic activity by performing simultaneous calcium imaging (using fluorescent calcium indicators) and voltage-sensitive dyes imaging. Our choice of calcium as an indicator of neuroglial activity is based on its recognized role as an important second messenger in multiple molecular signal transduction pathways, including those intimately involved in neurovascular communication. Finally, we will establish microscopic correlates of the large-scale hemodynamic and calcium signals by using 2-photon microscopy (Aim 3). Integration of macroscopic fMRI and calcium measurements with 2-photon data will allow a mechanistic interpretation of BOLD signals in terms of activity of underlying single cells and single blood vessels. PUBLIC HEALTH RELEVANCE: The relationship between functional MRI (fMRI) signals and the underlying microscopic neurovascular unit (NVU) physiology is a central unresolved issue in the interpretation of human functional neuroimaging data. The main goal of this proposal is to combine quantitative optical measurements of hemodynamic and neuroglial signals directly with BOLD fMRI aiming for a mechanistic interpretation of BOLD signals in terms of activity of the underlying single cells and single blood vessels. The proposed project will provide a mechanistic framework for understanding of fMRI signals and ultimately will guide the development of novel therapeutic, preventative, and diagnostic approaches.